Synthetic Proteins behind the Plasma Barrier: Molecular Spies

[Image: see text] There is a continuous demand to improve our understanding of fundamental processes that underlie human health and disease. Therefore, novel strategies that can assist in these efforts are required. For example, molecular biology and genetic approaches have revolutionized our understanding of protein-mediated processes by facilitating their direct visualization and analyses in living cells. Despite these developments, genetic manipulation has limitations in controlling events that occur after translation such as posttranslational modifications (PTMs), which are imperative regulatory elements. As a result, developing new methods to study PTMs in live cells is a major bottleneck in deciphering their exact roles in the myriad cellular processes. Synthetic and semisynthetic proteins are prepared by combining solid phase peptide synthesis (SPPS) and chemoselective ligation approaches with synthetic or recombinant peptides. Employing protein synthesis allows chemists to incorporate natural and unnatural modifications with virtually unlimited number of functional groups into the protein’s sequence, such as PTMs and their mimics. In addition, synthetic proteins can include additional elements such as fluorescent tags, reactive groups, caged units, and enrichment handles. Therefore, harnessing the power of chemical protein synthesis offers great opportunities to study fundamental biological processes. Unfortunately, the low cell permeability of proteins limits their applications mainly to in vitro settings, excluding live cell studies. As a result, chemical biologists have been attempting to overcome these limitations by developing protein delivery methods that would enable the study of custom-made proteins in a biological context. Success with these strategies should enable accurate determination of protein localization, degradation, folding, interactions, and involvement in the assembly of membrane-less organelles formed by liquid–liquid phase separation inside cells. Importantly, protein delivery approaches are complementary to genetic manipulations, and combining these approaches should pave the way to new discoveries. In this Account, we describe recent developments in protein delivery methods, with emphasis on those most compatible with synthetic proteins. We highlight experimental approaches and conceptual adaptations required to design and study synthetic proteins in live cells, with or without genetic manipulation. In addition, we highlight the strength and weakness of these approaches for both the delivery and the subsequent studies. We also describe our endeavors to deliver synthetic proteins to cells via cell penetrating peptides (CPPs) and multiplexed bead loading (MBL), as showcases of the applications of these methods to shed light on biological processes. Lastly, we contemplate other future applications of synthetic proteins to answer questions that are currently unapproachable.